UNAIDS has documented millions of new HIV-1 infections every year, thus a vaccine for HIV-1 is highly desirable. To date, despite numerous efforts, no immunization regimen reproducibly elicits broadly neutralizing antibodies (bNAbs) against HIV-1. Recently available native-like HIV-1 envelope spike (Env) trimers elicit antibodies that neutralize autologous tier-2 viruses but these antibodies have only limited potency and breadth. The observations that inferred germline (iGL) antibody precursors of bNAbs do not generally bind HIV-1 Env proteins or neutralizing HIV-1 suggested that rationally designed iGL-targeting Env immunogens would be required to initiate bNAb responses. Drs. Bjorkman and Nussenzweig propose a highly collaborative project to apply this approach to target epitopes of two classes of HIV-1 bNAbs: a class related to PGT121 that binds to the base of the V3 variable loop and interacts with the N332gp120 glycan (V3/N332 bNAbs), and IOMA-like bNAbs, a new class of CD4-mimetic CD4 binding site (CD4bs) bNAbs derived from the VH1-2 germline gene segment. The V3/N332 and IOMA classes were chosen because (i) V3/N332 Abs are among the most potent of bNAbs and are commonly found in HIV-infected individuals who develop bNAbs, (ii) IOMA's relatively low number of somatic hypermutations and its normal-length CDRL3 suggest it may be more easily elicited than VRC01-class VH1-2?derived CD4bs bNAbs that are heavily somatically mutated and contain rare 5-residue CDRL3s, and (iii) immunogen design will be facilitated the crystal structure of a natively-glycosylated Env trimer bound to the V3/N332 bNAb 10-1074 and to IOMA. The Bjorkman lab will create immunogens to target iGLs and shepherd bNAb maturation, while the Nussenzweig lab develops immunization schemes to elicit bNAbs using the designed immunogens. The Nussenzweig lab specific aims are: (1) Develop and simplify immunization protocols that elicit bNAbs to V3/N332 in iGL knock-in mice, (2) Adapt immunization protocols developed for V3/N332 bNAbs in knock-in mice to wild type and AlivaMab mice that carry un-rearranged human antibody loci, (3) Develop an immunization protocol to elicit IOMA-like antibodies, and (4) Determine the frequency of IOMA and V3/N332 bNAb precursors in the nave B cell repertoire of un-infected human donors. The Bjorkman lab specific aims are: (1) Solve structures of iGL?immunogen complexes to aid in structure-based immunogen design, (2) Use structure-based design and library screening to identify Env trimers that bind V3/N332 iGLs with high affinity, (3) Use structural information to guide construction of a yeast display library to find rare variants that bind IOMA iGL, (4) Combine results to create Env trimer immunogens that bind iGLs of both bNAbs and work with Dr. Nussenzweig to evaluate double and single immunogens in mice. These efforts will be supported by Core A (Automated cell/biochemical assays) and Core B (Protein Expression). The proposed experiments will produce candidate immunogens testing in macaques by collaborator Dr. Malcolm Martin and for vaccine trials in humans.